The B cell response to antigen is regulated by a variety of co-receptors that convey information to the B cell about the quality of the antigen and the status of the ongoing immune response. Over the last year we focused our attention of two potent regulators of B cell responses, namely the CD19/CD21 complex and the FcgammaRIIB. Signaling through the B cell receptor (BCR) is both amplified and prolonged by coligation of the BCR and the CD19/CD21 complex through the binding of complement fixed antigens. The low affinity Fc receptor, FcgammaRIIB, is a potent B cell inhibitory receptor and as such plays a central role in controlling antibody-mediated autoimmunity. Determining how these co-receptors influence BCR-induced signaling should add fundamentally to our understanding of the mechanism by which B cells are activated. Over the last year we have made progress in defining the mechanisms by which the positive coreceptor, the CD19/CD21 complex and the inhibitory receptor, FcgammaRIIB, function to regulate B cell signaling. [unreadable] [unreadable] Our earlier studies provided biochemical evidence that the B cell coreceptor, the CD19/CD21 complex, when coligated to the BCR through the binding of complement tagged antigens prolongs and enhances BCR signaling in part by prolonging the association of the BCR with sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts. We also provided biochemical and genetic evidence that the CD81 component of the CD19/CD21 complex was essential for the raft stabilizing function of the CD19/CD21 complex. We showed that CD81 associates with raft lipids upon coligation of the BCR and the CD19/CD21 complex and that in B cells from CD81-deficient mice coligated BCR and CD19/CD21 complexes failed to associate with raft lipids or enhance BCR signaling. We further demonstrated that upon coligation CD81 was palmitoylated and that the palmitoylation was essential for its raft-stabilizing function. Thus, we defined a novel mechanism by which a co-receptor influences the local lipid environment of a receptor namely by inducible lipidation. Using mutant CD81 in which the six cysteines that are modified by palmitoylation were changed to alanine, we demonstrated that palmitoylation was not necessary for interactions of CD81 with themselves but altered the pattern of expression on the surface and the downstream signaling from the BCR and CD19/CD21. We also provided biochemical evidence that the Fcgamma RIIB when coligated to the BCR destabilized the association of the BCR with lipid rafts. [unreadable] [unreadable] In these studies lipid rafts were operationally defined by their relative detergent insolubility, due to the tight packing of the saturated chains of the raft lipids and by their dependence on cholesterol. However, the use of detergents and cholesterol-depleting drugs are fraught with potential artifacts including creating the lipid heterogeneities we set out to study. High resolution fluorescence resonance energy transfer (FRET) coupled with total internal reflection microscopy (TIRFM) or confocal microscopy offered the opportunity to quantify the interactions of the BCR with raft lipids in live cells over the time and length scale necessary to capture the earliest events in antigen-initiated B cell activation. [unreadable] [unreadable] To study the interactions of raft lipids with BCRs we generated cell lines that expressed a BCR containing the FRET donor fluorescent protein CFP and the FRET acceptor protein, YFP, tethered to the membrane by either raft lipids or by non-raft lipids. FRET confocal imaging of living B cells revealed that within seconds of antigen binding the BCR selectively and transiently associated with the lipid raft constructs and that this association was prolonged by coengagement of the BCR and the CD19/CD21 coreceptor complex. Using live cell FRET TIRF imaging we recently provided direct evidence that the association of the BCR with raft lipids following antigen binding is blocked by the FcgammaRIIB. Thus, these FRET measurements provided the first direct evidence for the antigen-induced association of the BCR with lipid rafts in living cells and the regulation of this very early event by the CD19/CD21 complex and the FcgammaRIIB. [unreadable] [unreadable] We have also initiated studies to describe the interactions of the BCR and FcgammaRIIB during B cell activation by TIRF microscopy. Surprisingly, we observed that the binding of antigen to the BCR resulted in the recruitment of the FcgammaRIIB to the clustered BCRs. We are interested in determining both the function of the recruitment and the underlying mechanism. [unreadable] [unreadable] In addition to the well studied inhibitory pathway initiated by crosslinking the BCR and FcgammaRIIB, we provided evidence that when clustered independently of the BCR, the FcgammaRIIB initiates an ITIM-, Lyn- and SHIP-independent pathway that triggers apoptosis through a mechanism that involves c-Abl family kinases. Thus, the FcgammaRIIB has the ability to block the BCR-dependent, antigen-driven activation of B cells as well as antigen-independent, BCR-independent B cell activation. It was recently shown in mice that long lived bone marrow plasma cells express the FcgammaRIIB and that engaging the FcgammaRIIB alone by immune complexes induced these plasma cells to undergo antigen-independent apoptosis. Over the last year we provided evidence in humans that the FcgammaRIIB functions independently of the BCR to inhibit plasma cells and naive B cells but not memory B cells. These results suggest that the BCR-independent FcgammaRIIB signaling pathway may play an important role in humans in acutely controlling antibody levels by inhibiting antibody secreting PCs and the activation of naive B cells without affecting the long-lived memory B cell pool.